JP4087471B2 - Measuring chip for surface plasmon resonance biosensor and manufacturing method thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a measuring chip for a surface plasmon resonance biosensor and a manufacturing method thereof.
[0002]
[Prior art]
At present, many measurements using immune responses are performed in clinical tests, etc., but the conventional method requires complicated operations and labeling substances, so it is highly sensitive to changes in the ligand without the need for labeling substances. An immunosensor using surface plasmon resonance (SPR) that can be detected in a short time has been used.
[0003]
A measurement chip generally used in such a measurement apparatus (surface plasmon resonance biosensor) using surface plasmon resonance has a structure as shown in FIG. That is, a porous material 4 is formed on a metal film 2 ′ formed on a glass substrate 1 ′, and a physiologically active substance such as an enzyme or an antibody is carried on the surface and inside of the porous material 4. Or it is fixed. As the porous material 4, for example, a woven fabric, a knitted fabric, a non-woven fabric, a porous inorganic or organic material made of synthetic fiber, natural fiber, inorganic fiber, or the like is used (see JP-A-3-164195). In addition, carboxymethyldextran is used as the porous material 4 in a commercial product (for BIAcore 2000, manufactured by Pharmacia Biosensor).
[0004]
However, since the only physiologically active substances that interact with the measurement object substantially and efficiently are those present on the surface of the porous material 4, the physiologically supported or fixed inside the porous material 4 is used. The active substance does not function effectively, and the sensitivity decreases accordingly.
[0005]
In addition, as a method for fixing the physiologically active substance to the metal film 2 ′, there is a case where the LB (Langmuir-Blodgett) method is used (see Japanese Patent Laid-Open No. 5-288672). There is a problem that the LB film falls off together with the physiologically active substance.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a measurement chip for a surface plasmon resonance biosensor that can obtain good sensitivity and can be easily manufactured even when a small amount of physiologically active substance is immobilized.
[0007]
[Means for Solving the Problems]
As a result of diligent research in view of the above problems, the present inventor has formed an organic sulfur layer on a metal film and immobilized a physiologically active substance on the organic sulfur layer, even if a small amount of physiologically active substance is used. The inventors found that good sensitivity can be obtained and completed the present invention.
That is, the present invention is a measurement chip for a surface plasmon resonance biosensor comprising a transparent substrate, a metal film disposed on the transparent substrate, and an organic sulfur layer disposed on the metal film. is there.
[0008]
Moreover, this invention is a manufacturing method of the measuring chip for surface plasmon resonance biosensors which arrange | positions an organic sulfur layer on this metal film after arrange | positioning a metal film on a transparent substrate.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The measurement chip for surface plasmon resonance biosensor in the present invention (hereinafter simply referred to as “measurement chip”) is a chip used for a surface plasmon resonance biosensor, and transmits and reflects light emitted from the sensor. This means a member including a part and a part for fixing a physiologically active substance, and may be fixed to the main body of the sensor or may be removable.
[0010]
The measurement chip of the present invention includes a transparent substrate, a metal film disposed on the transparent substrate, and an organic sulfur layer disposed on the metal film. Here, “a metal film disposed on a transparent substrate” refers to a case in which a metal film is disposed on a transparent substrate in direct contact with another layer without directly contacting the transparent substrate. It is meant to include the case where they are arranged via. The “organic sulfur layer disposed on the metal film” has the same meaning as described above.
[0011]
FIG. 2 shows a schematic cross-sectional view of a measuring chip according to an example of the present invention.
The measurement chip according to the present example has a transparent substrate 1, a metal film 2 formed on the transparent substrate 1, and an organic sulfur layer 3 formed on the metal film 2.
The transparent substrate 1 may be any material as long as it is normally used for a measurement chip for a surface plasmon resonance biosensor, and is generally transparent to laser light such as glass, polyethylene terephthalate, and polycarbonate. A material made of a material can be used, and a material that does not exhibit anisotropy with respect to polarized light and has excellent workability is desirable, and its thickness is about 0.1 to 20 mm.
[0012]
The metal film 2 is not particularly limited as long as surface plasmon resonance can occur. Gold, silver, copper, aluminum, platinum, etc. are mentioned as a kind of metal which can be used for this metal film 2, These can be used individually or in combination. In consideration of adhesion to the transparent substrate 1, an intervening layer made of chromium or the like may be provided between the transparent substrate 1 and a layer made of gold, silver or the like.
[0013]
The film thickness of the metal film 2 is preferably 100 to 2000 mm, and particularly preferably 200 to 600 mm. If it exceeds 3000 mm, the surface plasmon phenomenon of the medium cannot be detected sufficiently. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 5-50 mm.
[0014]
The metal film 2 may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like. Among these methods, it is preferable to use a sputtering method.
[0015]
The organic sulfur layer 3 is a layer made of a substance having at least one organic functional group bonded to a metal atom in the metal film 2 through a sulfur atom. The thickness of the organic sulfur layer 3 is preferably 10 to 200 mm, and particularly preferably 10 to 50 mm.
[0016]
The organic sulfur layer 3 can be formed using a compound having a mercapto group and another functional group (hereinafter simply referred to as “thiol compound”). Here, as a preferable thiol compound, the compound represented by the following formula (1), formula (2), formula (3), formula (4) or formula (5) can be exemplified.
[0017]
Equation _{(1): HS- (CH 2} ) n -NH 2
Equation _{(2): HS- (CH 2} ) n -COOH
Equation _{(3): HS- (CH 2} ) n -C (NH 2) 3
Equation _{(4): HS- (CH 2} ) n -CHO
Equation _{(5): HS- (CH 2} ) n -SH
[0018]
Among these compounds, it is preferable to use a compound represented by the formula (3). This is because this compound has a plurality of amino groups in one molecule, so that more physiologically active substances can be immobilized.
[0019]
Specific examples of the compound represented by the formula (1) include mercaptoaminomethane, 2-mercapto-1-aminoethane, 3-mercapto-1-aminopropane, and 4-mercapto-1-aminobutane. Specific examples of the compound represented by the formula (2) include mercaptoacetic acid, 2-mercaptopropionic acid, 3-mercaptobutyric acid, 4-mercaptovaleric acid, and the like, which are represented by the formula (3). Specific examples of the compound include 1,1,1-triamino-2-mercaptoethane, 1,1,1-triamino-3-mercaptopropane, and the like represented by the formula (4) Examples of such compounds include mercaptoacetaldehyde, 2-mercaptopropyl aldehyde, 3-mercaptobutyraldehyde, 4-mercapto barrel aldehyde. Specific examples of the compound represented by the formula (5) include dimercaptomethane, 1,2-dimercaptoethane, 1,3-dimercaptopropane, and 1,4-dimercaptobutane. 1,5-dimercaptopentane and the like.
[0020]
As a method for forming an organic sulfur layer using a thiol compound, a method of exposing the metal film 2 to a saturated vapor of a thiol compound for a certain time (saturated vapor method), or a method of exposing the metal film 2 to a solution containing a thiol compound for a certain time. Examples include a dipping method (dipping method), a method using a spin coater (spin coating method), a method using a gravure printing machine (gravure method), and the like.
[0021]
In addition, a silane coupling agent can form a molecular layer having an organic functional group on the metal film 2 similarly to the thiol compound, but a silane coupling agent cannot form a molecular layer having a carboxyl group. . Therefore, the thiol compound is particularly useful when introducing a carboxyl group.
[0022]
The organic sulfur layer 3 in the present invention has the following advantages.
{Circle around (1)} Since the physiologically active substance can be immobilized at a position very close to the metal film 2, the measurement sensitivity can be greatly improved as compared with the case where a conventional measurement chip is used.
(2) Film formation is easy, and a large amount of film formation can be performed at one time.
(3) By changing the type of thiol compound, not only film thickness but also chemical modification such as surface modification and functional group introduction becomes possible.
In the measurement chip of the present invention, a physiologically active substance is fixed to the organic sulfur layer 3 directly or via a water-soluble polyvalent reagent.
[0023]
The physiologically active substance is not particularly limited as long as it interacts with the measurement target, and examples thereof include immune proteins, enzymes, microorganisms, and nucleic acids. Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.
[0024]
The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme and the like can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. Also, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit a specific reaction with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.
[0025]
The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA or RNA can be used.
The thickness of the physiologically active substance depends on the size of the physiologically active substance itself used, but is preferably 100 to 3000 mm, particularly preferably 100 to 1000 mm.
[0026]
When the physiologically active substance is a protein such as an antibody or an enzyme, the immobilization can be performed by bringing a predetermined amount of the physiologically active substance into contact with the organic sulfur layer 3 for a predetermined time. For example, it can be immobilized by installing a measurement chip on a flow cell type surface plasmon resonance biosensor and allowing a physiologically active substance at a constant flow rate to flow for a predetermined time (a predetermined amount).
[0027]
When the physiologically active substance is a nucleic acid, the immobilization is performed by binding avidin 51 to an organic functional group on the organic sulfur layer 3 and binding a nucleic acid 53 labeled with biotin 52 to the avidin 51 as shown in FIG. This can be done. As a method for labeling the nucleic acid 53 with biotin 52, a method of performing PCR using a primer to which biotin 52 is bound can be exemplified.
[0028]
The water-soluble polyvalent reagent is not particularly limited as long as the physiologically active substance can be firmly immobilized covalently, and may be used according to the organic functional group on the organic sulfur layer 3. For example, if the organic functional group on the organic sulfur layer 3 is an amino group, cysteine, glutaraldehyde, periodic acid, N, N′-o-phenylene dimaleimide, N-succinimidyl-4- (N-maleimidomethyl) Cyclohexane-1-carboxylate, N-succinimidylmaleimidoacetic acid, N-succinimidyl-4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid, N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1 -Carboxylic acid, N-sulfosuccinimidyl-3-maleimidobenzoic acid, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N -(8-maleimidocapryloxy) sulfosuccinimide Sodium salt, N- (11-maleimidoundecanoyloxy) sulfosuccinimide / sodium salt, N [2- (1-piperazinyl) ethyl] maleimide / maleimide compound exemplified by dihydrochloride and N-succinicidylpyridyldithiocarboxylate And when the organic functional group is a mercapto group, cysteine, N, N′-o-phenylene dimaleimide, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl maleimide acetic acid, N-succinimidyl-4-maleimidobutyric acid, N-succinimidyl-6-maleimidohexanoic acid, N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid, N-sulfo Succinimidyl-3-maleimidobenzoic acid, -(4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11- Maleimide undecanoyloxy) sulfosuccinimide / sodium salt, N [2- (1-piperazinyl) ethyl] maleimide / maleimide compound exemplified by dihydrochloric acid, N-succinicidylpyridyldithiocarboxylate, and the like can be used, If the organic functional group is a carboxyl group and an aldehyde group, cysteine, oxalic acid hydrazide, succinic acid hydrazide, adipic acid hydrazide, sebacic acid hydrazide, oxalic acid dihydrazide, succinic acid dihydrazide, adipic acid dihydrazide, seba For example, but not limited to diacid hydrazide can be used.
[0029]
Since the physiologically active substance is firmly immobilized through the water-soluble polyvalent reagent in this manner, the physiologically active substance can be immobilized even when the measurement chip is washed, and therefore it can be used for repeated measurement. The advantage that it can be obtained.
[0030]
The water-soluble polyvalent reagent can be immobilized on the organic sulfur layer 3 by bringing a predetermined amount of the water-soluble polyvalent reagent into contact with the organic sulfur layer 3 for a predetermined time. For example, it can be immobilized by installing a measurement chip on a flow cell type surface plasmon resonance biosensor and flowing a water-soluble polyvalent reagent at a constant flow rate for a predetermined time (predetermined amount). The physiologically active substance can be immobilized on the water-soluble polyvalent reagent in the same manner as when the physiologically active substance is immobilized on the organic sulfur layer 3.
[0031]
The measurement chip of the present invention can be used for, for example, a surface plasmon resonance biosensor as shown in FIG.
This surface plasmon resonance biosensor has a cartridge block 7, a light source 8, and a detector 9, and the measurement chip 6 of the present invention is installed on the cartridge block 7 for use. The measuring chip 6 is installed so that the transparent substrate is on top. A concave portion is provided on the upper surface of the cartridge block 7, and the concave portion and the measurement chip 6 constitute a measurement cell 71. The measurement cell 71 communicates with the outside of the cartridge block 7 through flow paths 72 and 73, and the sample flows into the measurement cell 71 through the flow path 72 and is discharged to the outside through the flow path 73 after being used for measurement. .
[0032]
Monochromatic light is irradiated from the light source 8 toward the transparent substrate of the measuring chip 6 (incident light 80), and the reflected light 90 reflected by the metal film provided on the back surface of the measuring chip 6 enters the detector 9. Shine. The detector 9 can detect the intensity of the reflected light 90.
[0033]
With the above structure, a reflected light intensity curve that forms a valley with respect to a certain incident angle θ is obtained. The valleys in the reflected light intensity curve are due to surface plasmon resonance. That is, when light is totally reflected at the interface between the transparent substrate of the measuring chip 6 and the outside, surface waves called evanescent waves are generated at the interface, while surface waves called surface plasmons are also generated at the metal film. When the wave numbers of the two surface waves coincide with each other, resonance occurs, and a part of the energy of light is used to excite surface plasmons, and the intensity of reflected light is reduced. Here, the wave number of the surface plasmon is affected by the refractive index of the medium very close to the surface of the metal film. Therefore, if the refractive index of the medium changes due to the interaction between the measurement target substance and the physiologically active substance, the surface plasmon resonance The incident angle θ at which is generated changes. Therefore, it is possible to detect a change in the concentration of the measurement target substance based on the deviation of the valley of the reflected light intensity curve. The change amount of the incident angle θ is called a resonance signal, and a change of 10 ⁻⁴ ° is expressed as 1 RU.
[0034]
【The invention's effect】
The measurement chip of the present invention is easy to manufacture and can measure a measurement target substance with good sensitivity even if a small amount of physiologically active substance is immobilized.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view of a conventional measuring chip.
FIG. 2 is a schematic cross-sectional view of one embodiment of a measuring chip of the present invention.
FIG. 3 is a schematic cross-sectional view of one embodiment of a measurement chip of the present invention on which a nucleic acid is immobilized.
FIG. 4 is a conceptual diagram of a surface plasmon resonance biosensor used in the measurement chip of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1,1 '... Transparent substrate 2, 2' ... Metal film 3 ... Organic sulfur layer 4 ... Porous material
51 ... Avidin
52 ... Biotin
53 ... Nucleic acid 6 ... Measurement chip 7 ... Cartridge block
71… Measurement cell
72, 73 ... flow path 8 ... light source
80 ... Incident light 9 ... Detector
90 ... Reflected light

Claims (6)

A transparent substrate, a metal film disposed on the transparent substrate, and an organic sulfur layer having a thickness of 10 to 200 mm disposed on the metal film, the following formula: HS— (CH ₂ ) _n —C (NH ₂ ) ₃
A measurement chip for a surface plasmon resonance biosensor, comprising an organic sulfur layer containing a compound represented by the formula:   The measurement chip for a surface plasmon resonance biosensor according to claim 1, wherein the organic sulfur layer includes, in addition to the compound represented by the above formula, other compounds having a mercapto group and another functional group. . The other compound having the mercapto group and another functional group is represented by the following formula (1), formula (2), formula (4) or formula (5);
Equation _{(1): HS- (CH 2} ) n -NH 2
Equation _{(2): HS- (CH 2} ) n -COOH
Equation _{(4): HS- (CH 2} ) n -CHO
Equation _{(5): HS- (CH 2} ) n -SH
The measurement chip for a surface plasmon resonance biosensor according to claim 2, wherein the measurement chip is a compound represented by the formula: After placing the metal film on a transparent substrate, wherein the following equation on the metal _{film: HS- (CH 2) n -C} (NH 2) 3
A method for producing a measurement chip for a surface plasmon resonance biosensor, characterized in that an organic sulfur layer containing a compound represented by the formula:   5. The surface plasmon resonance biosensor according to claim 4, wherein the organic sulfur layer is formed using another compound having a mercapto group and another functional group in addition to the compound represented by the above formula. Method for manufacturing measuring chip. The other compound having the mercapto group and another functional group is represented by the following formula (1), formula (2), formula (4) or formula (5);
Equation _{(1): HS- (CH 2} ) n -NH 2
Equation _{(2): HS- (CH 2} ) n -COOH
Equation _{(4): HS- (CH 2} ) n -CHO
Equation _{(5): HS- (CH 2} ) n -SH
The method for producing a measurement chip for a surface plasmon resonance biosensor according to claim 5, wherein the compound is represented by the formula:

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